Many growth factors and hormones such as nerve growth factor (NGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF) an insulin mediate their signals through interactions witi cell surface tyrosine kinase receptors. The transduction of extracellular signals across the membrane, initiated by ligand binding, leads to the propagation of multiple signaling events which ultimately control target biochemical pathways within the cell.
The phosphatidylinositol 3-kinases (PI3 kinases or PI3Ks) represent a ubiquitous family of heterodimeric lipid kinases that are found in association with the cytoplasmic domain of hormone and growth factor receptors and oncogene products. PI3Ks act as downstream effectors of these receptors, are recruited upon receptor stimulation and mediate the activation of second messenger signaling pathways through the production of phosphorylated derivatives of inositol. PI3Ks phosphorylate phosphatidylinositol (PtdIns) at the 3'-hydroxyl of the inositol ring and substrates include PtdIns, PtIns(4)phosphate PtdIns(4,5)bisphosphate and PtdIns(3,4,5) triphosphate with the major product being PtdIns(3,4,5)triphosphate (Fry, Biochim. Biophys. Acta., 1994, 1226, 237-268).
PI3Ks have been implicated in many cellular activities including growth factor mediated cell transformation, mitogenesis, protein trafficking, cell survival and proliferation, DNA synthesis, apoptosis, neurite outgrowth and insulin-stimulated glucose transport reviewed in (Fry, Biochim. Biophys. Acta., 1994, 1226, 237-268).
The PI3 kinase enzyme heterodimers consist of a 110 kD (p110) catalytic subunit associated with an 85 kD (p85) regulatory subunit and it is through the SH2 domains of the p85 subunit that the enzyme associates with the membrane-bound receptors. Multiple isocforms of the p110 catalytic subunit have been reported and they have been divided into three classes, IA and IB, II, and III. Class IA includes the alpha, beta and delta isoforms all of which bind to p85 and associate with receptor tyrosine kinases (Hu et al., Mol. Cell. Bio., 1993, 13, 7677-7688: Stirdivant et al., Bioorg. Med. Chem., 1997, 5, 65-74; Vanhaesebroeck et al., Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 4330-4335). While the alpha and beta isoforms are ubiquitously expressed, the delta isoform is found only in white blood cells (Vanhaesebroeck et al., Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 4330-4335). Class IB includes the gamma isoform of p110 which exhibits, no p85 binding, is regulated by the .alpha. and .beta..gamma. subunits of G-proteins, and contains unique C2 domains (Stoyanov et al., Science, 1995, 269, 690-693). Classes II and III contain the cpk and Vps34p isoforms, respectively (Molz et al., J. Biol. Chem., 1996, 271, 13892-13899; Schu et al., Science, 1993, 260, 88-91).
PI3 kinase p110 delta is the most recent catalytic isoform to be isolated (Vanhaesebroeck et al., Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 4330-4335). Unlike other members of the class IA isoforms, the delta isoform has distinct biochemical and structural properties. For example, it does not phosphorylate the p85 subunit but instead contains an intrinsic autophosphorylation capacity. In addition the delta isoform has a restricted pattern of expression, that being to white blood cells. A potential role for PI3 kinase p110 delta in cytokine signaling is suggested by its localization to subpopulations of these cells involved in migration. Investigations of the coupling of the delta isoform to receptor complexes in myeloid and lymphoid cell lines revealed that PI3 kinase p110 delta may play a role in leukocyte transendtothelial migration (Vanhaesebroeck et al., Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 4330-4335).
Other studies implicate the PI3 kinase p110 delta isoform in nitric oxide signaling. It has been demonstrated that nitrosative and oxidative stressors can trigger ras- and PI3 kinase-regulated events in the cell. Studies of redox signaling in human T cells showed that only the beta and delta isoforms of PI3 kinase p110 were recruited to the membrane upon free radical generation from nitric oxide donors (Deora et al., J. Biol. Chem., 1998, 273, 29923-29928). These findings suggest a role for PI3 kinase beta in inflammatory conditions such as rheumatoid arthritis and asthma.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of PI3 kinase p110 delta.
To date, strategies aimed at inhibiting PI3 kinase p110 delta function have involved the use of antibodies and chemical inhibitors.
Several chemically distinct inhibitors for PI3 kinases are reported in the literature. These include wortmannin, a fungal metabolite (Ui et al., Trends Biochem. Sci., 1995, 20, 303-307); demethoxyviridin, an antifungal agent (Woscholski et al., FEBS Lett., 1994, 342, 109-114) and quercetin and LY294002, two related chromones (Vlahos et al., J. Biol. Chem., 1994, 269, 5241-5248). However, these strategies are untested as therapeutic protocols as well as being non-specific to the PI3 kinase p110 delta isoform. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting PI3 kinzise p110 delta function.
Antisense technology is emergling as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of PI3 kinase p110 delta expression.